Influence of the polyunsaturated fatty acids linoleic acid, arachidonic acid, α -linolenic acid and γ -linolenic acid on melanogenesis
of B16 mouse melanoma cells and normal human melanocytes
INAUGURALDISSERTATION
zur
Erlangung der Würde eines Doktors der Philosophie vorgelegt der
Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel
von
Martin Stöckli aus Hofstetten (SO)
Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von
Prof Dr. Jürg Meier und PD Dr. Reto Brun
Basel, den 12. Februar 2002
Prof. Dr. Andreas D. Zuberbühler Dekan
Dedicated to my wife Marlene
CoConntteennttss
CONTENTS ... 1
ABBREVIATIONS... 4
SUMMARY ... 6
1. INTRODUCTION ... 7
1.1 The skin ... 7
1.2 Melanocytes and melanogenesis... 10
1.2.1 Melanocytes ... 11
1.2.2 Melanin biosynthesis ... 14
1.2.3 Regulation of melanogenesis ... 16
1.2.3.1 Enzymes in skin pigmentation... 17
1.2.3.2 Paracrine control ... 20
1.2.3.3 Hormonal control... 21
1.2.3.4 Transmembrane and intracellular pathways ... 24
1.2.3.5 Effect of UV radiation... 26
1.3 Fatty acids ... 27
1.3.1 Nomenclature of fatty acids ... 27
1.3.2 Biosynthesis of polyunsaturated fatty acids ... 28
1.3.3 PUFA in cutaneous biology ... 28
1.4 PUFA and melanogenesis ... 30
1.6 Objectives for the thesis... 31
2. MATERIAL AND METHODS ... 33
2.1. Cell culture ... 33
2.1.1 Isolation and cultivation of normal human melanocytes... 33
2.1.2 Elimination of fibroblast contamination ... 35
2.1.3 Characterization of melanocytes... 36
2.1.4 Cultivation of B16-F1 and B16-F10 mouse melanoma cells ... 38
2.1.5 Determination of cell number... 39
2.2 Test substances ... 40
2.3 Determination of cytotoxicity... 40
2.3.1 Cytotoxicity on normal human melanocytes... 41
2.3.2 Cytotoxicity on B16 mouse melanoma cells ... 43
2.4 Cell culture and assay conditions ... 45
2.4.1 Mouse melanoma cells ... 45
2.4.1.1 Melanin content and tyrosinase activity... 45
2.4.1.2 Determination of whole melanin... 46
2.4.1.3 Tyrosinase content ... 46
2.4.1.4 Expression of tyrosinase gene... 47
2.4.2 Normal human melanocytes ... 47
2.4.2.1 Melanin content... 47
2.4.2.2 Tyrosinase activity ... 47
2.4.2.3 Tyrosinase content ... 48
2.4.2.4 Expression of tyrosinase gene... 49
2.5 Determination of melanin content ... 49
2.5.1 Method according to Ando... 49
2.5.2 Method according to Nakajima... 50
2.5.3 Method according to Bathnagar ... 50
2.6 Determination of tyrosinase activity... 51
2.6.1 Method according to Ando... 51
2.6.2 MBTH-method ... 51
2.7 Quantitative analysis of tyrosinase content of cell lysates by ELISA... 52
2.7.1 Quantification of mouse tyrosinase... 53
2.7.1.1 Solutions and buffers ... 53
2.7.1.2 Determination of optimum of conjugate concentration ... 54
2.7.1.3 Determination of optimum concentration of control peptide and anti tyrosinase antibody55 2.7.1.4 Specificity of the ELISA ... 57
2.7.1.5 ELISA for the quantification of mouse tyrosinase ... 59
2.7.2 Quantification of human tyrosinase ... 60
2.7.2.1 Solutions and buffers ... 61
2.7.2.2 Specificity of the ELISA ... 62
2.7.2.3 ELISA for the quantification of human tyrosinase... 62
2.8 Quantitative analysis of tyrosinase content of cell lysates by Western Blot ... 62
2.8.1 SDS-PAGE ... 62
2.8.2 Blotting... 63
2.9 Tyrosinase gene expression ... 65
2.9.1 Isolation of total RNA from cells ... 66
2.9.2 Synthesis of cDNA ... 68
2.9.3 Quantitative real-time TaqMan PCR ... 68
2.10. Influence of PUFA on melanogenesis of MelanoDerm... 71
3. RESULTS ... 72
3.1.Cell culture ... 72
3.1.1 Normal human melanocytes ... 72
3.1.2 Mouse melanoma cells ... 74
3.2 Cytotoxicity of PUFA... 76
3.2.1 Cytotoxicity on normal human melanocytes... 76
3.2.2 Cytotoxicity on mouse melanoma cells... 77
3.3 Effects of PUFA on melanogenesis of mouse melanoma cells... 79
3.3.1 Tyrosinase mRNA content... 79
3.3.2 Tyrosinase activity ... 80
3.3.3 Melanin content... 82
3.3.4 Tyrosinase content ... 86
3.4 Effects of PUFA on melanogenesis of normal human melanocytes ... 89
3.4.1 Tyrosinase mRNA content... 89
3.4.2 Tyrosinase activity ... 90
3.4.3 Melanin content... 91
3.4.4 Tyrosinase content ... 92
3.5 Effects of PUFA on pigmentation of MelanoDerm... 94
4. DISCUSSION ... 98
4.1 B16 mouse melanoma cells ... 98
4.2 Normal human melanocytes... 104
4.3 Concluding remarks... 106
5. REFERENCES ... 108
6. ACKNOWLEDGEMENTS ... 115
7. CURRICULUM VITAE ... 116
AbAbbbrreevviiaattiioonnss
AA Arachidonic acid
ACTH Adrenocorticotrophic hormone ASP Agouti signaling protein
bFGF Basic fibroblast growth factor
cAMP 3’,5’-cyclic adenosine monophosphate DABCO 1,4-Diazabicyclo[2.2.2]octan DHI Dihydroxyindole
DHICA 5,6-Dihydroxyindole-2-carboxylic acid DOPA 3,4-Dihydroxyphenylalanine
ET-x Endothelin x (e. g. ET-1)
GAPDH Glyceraldehyde-3-phosphate Dehydrogenase GTP Guanosine 5’-triphosphate
HGF Hepatocyte growth factor
IBMX 3-Isobutyl-1-methylxanthine ICAM-1 Intercellular adhesion molecule 1 IL-x Interleukin x (e. g. IL-1)
INF-γ Interferon-γ
IP3 Inositol 1,4,5-trisphosphate
kDa Kilo Dalton
LA Linoleic acid
α-LA α-Linolenic acid γ-LA γ-Linolenic acid
MGF Mast cell growth factor
MHC Major histocompatibility complex
MITF Microphtalmia associated transcriprion factor MSH Melanocyte stimulating hormone
NHEK Normal human epidermal keratinocytes NHM Normal human melanocytes
PIP2 Phosphatidylinositol 4,5-bisphosphate
PKA cAMP dependent protein kinase PKC-β Protein kinase-β
PLC Phospholipase C
PMA Phorbol-12-myristate-13-acetate POMC Pro-opiomelanocortin
PUFA Polyunsaturated fatty acid TNF-α Tumor necrosis factor-α
TRP1 Tyrosinase related protein 1 TRP2 Tyrosinase related protein 2 WFI Water for injection
SuSummmmaarryy
The influence of the polyunsaturated fatty acids (PUFA) linoleic acid, arachidonic acid, α-linolenic acid and γ-linolenic acid on pigmentation was examined using different in vitro models: monolayer cultures of B16 mouse melanoma cells, monolayer cultures of normal human melanocy- tes and a commercially available reconstructed pigmented epidermis that consists of normal, human-derived epidermal keratinocytes (NHEK) and melanocytes (NHM) which have been cultured to form a multilaye- red, highly differentiated model of the human epidermis that exhibits in vivo-like morphological and ultrastructural characteristics.
We used two different sublines of B16 origin called B16-F1 and B16-F10 for our experiments. In both sublines 25 µM of the PUFA listed above showed two distinct effects on melanogenesis of the cells: 1) the secre- tion of intracellularly produced melanin into the extracellular culture fluid was enhanced, and 2) the intracellular melanin content was dec- reased. The stimulation of melanin secretion was highest after an incu- bation time of 24 h, whereas the reduction of intracellular melanin was most effective after 72 h. α-Linolenic acid and γ-linolenic acid decrea- sed intracellular melanin content more effectively than arachidonic acid and linoleic acid. Futhermore intracellular melanin content of murine melanoma cells was reduced more effectively in B16-F10 subline.
In both sublines tyrosinase activity was not affected, whereas tyrosina- se content was reduced. Tyrosinase mRNA level of B16-F1 cells was decreased by linoleic and arachidonic acid. None of the fatty acids had an influence on tyrosinase mRNA levels of B16-F10 cells.
None of the polyunsaturated fatty acids at a concentration of 100 µM affected the melanogenesis of monolayer cultures of NHM when measu- ring the changes in mRNA level, content and activity of tyrosinase and in the amount of intracellular pigment. In addition the polyunsaturated fatty acids did not inhibit the pigmentation of the reconstructed pig- mented epidermis.
1.1. IInnttrroodduuccttiioonn 1.1.11 TThhee sskkiinn
The skin has many functions: it not only protects the body against mechanical, thermic and chemical influences, but is also a highly sensitive organ for communication. Although the skin has only a thickness of a few millimeters, it is, with a surface of 1.8 m2 and an average weight of 4.8 kilograms, one of the biggest organs of the human body. The skin represents an effective barrier and protects the remaining organs against mechanical, physical, chemical and bio- logical damage. Furthermore, the skin possesses a crucial function in heat exchange and protection from loss of water. This transepidermal water loss is about 0.2 to 0.4 mg/cm2/h at 30°C compared to the rate of evaporation of water from a free, uncovered water surface that is about 35 mg/cm2/h at 30°C. The skin also has important endocrine functions such as the synthesis of vitamin D3, sex hormones and pheromones and it provides also immunological defenses.
Fig. 1.1 Structure of the skin
The skin is divided in to three layers: the epidermis (1), the dermis (2) and the hypodermis (3). The epidermis is a renewing stra tified epithelium. The dermis, that co ns is ts of ex t ra cellula r ma trix (co lla gen, elas tic fi- bers, etc.) and inters persed cells such as fibroblasts, is well supplied with blood vessels. There are no distinct borders between the dermis and the hypodermis that consis ts of fat cells (a dipocytes).
(From “Elementa dermatologica”; Christophers E., Sterry W., Schubert Ch., Br äuer H.; Ca ssella-Riedel Pharma Frankfurt, 1987)
The skin is divided in three layers (Fig. 1.1): the epidermis arising from the ectoderm, the dermis and the hypodermis which are of mesodermal origin.
The epidermis is the outermost part of the skin. It is a continually renewing, laminated, squamous epithelium (Fig. 1.2). The main cells of the epidermis are the so-called keratinocytes. Beside the keratino- cytes there are lymphocytes, Langerhans cells, melanocytes and Merkel cells interspersed among them. The keratinocytes are ar- ranged in layers, which represent the different stages of their differ- entiation.
Fig. 1.2 Structure of epidermis and underlying dermis
The epidermis cons is ts of layers of keratino cytes (a-d) that are named as fo llows:
s t ratum ba sa le (a ), stra tum spinosum (b), stra tum granulosum (c) and stra tum corneum (d).
In the stratum basale pigment cells, the so-called melanocytes, are found (1).
The dermis is well supported by blood vessels (2) and cons ists of an extracellular matrix made of an amorphous ground substance and fibrillar structures such as collagen bundles (4). The dermis is a product of specialized cells called fibroblasts (3).
(From “Elementa dermatologica”;
Christophers E., St erry W., Schu- bert Ch., Bräuer H.; Cassella- Riedel Pharma Frankfurt, 1987)
b
a d c
2 1
3
4
The layers are named either according to their function or to their structure; their names are:
• Basal layer or stratum germinativum
• Spinous layer or stratum spinosum
• Granular layer or stratum granulosum
• Transitional layer or stratum lucidum
• Horny layer or stratum corneum
Some of the basal keratinocytes in the stratum germinativum are mi- totically active and give rise to cells that move up to the more super- ficial layers. The stratum germinativum is followed by one or more layers of larger keratinocytes which are connected by desmosomes.
This is the so called stratum spinosum. Its name derives from the spine-like appearance of the cells that result from shrinking during the processing of tissues for histological analyses.
In the stratum granulosum basophlic granulae can be seen in the light microscope, which contain keratohyalin, a precursor of keratin.
Above the stratum granulosum there is the stratum lucidum, which can be found only in very strongly cornified and hairless skin.
The stratum corneum is made of flattened epithelial cells (corneo- cytes) arranged in multiple layers. These layers are called keratinized layers because of the synthesis of the protein keratin in those cells.
Keratin is a structure protein that is specific to the skin, hair and nails.
This layer of skin is, for the most part, dead. Acidic and basic kera- tins make up about 80% of the dry mass of the corneocytes.
It takes about one month from the time a basal cell leaves the bot- tom layer until it is desquamated.
The differentiation of keratinocytes includes several steps:
• Synthesis and modification of structural proteins, especially kera- tins
• Appearance of new organelles, reorganization of existing organ- elles, and loss of organelles
• Change in cell size and shape
• Specialization of cellular metabolism
• Changes in the properties of cell membranes
• Dehydration
The stratum corneum is an effective barrier against water loss and is mostly impermeable to external substances, such as drugs and toxic materials.
The dermis consists of extracellular matrix (collagen, elastic fibers, filamentous structures and amorphous ground substances made of glycosaminoglycans) that is interspersed with fibroblasts, macro- phages, mast cells and lymphocytes.
The hypodermis consists of three fatty layers separated by connec- tive tissue sheets. The primary functions of the hypodermis are ther- moregulation, cushioning against mechanical trauma, contouring the body, filling space, and, the most important, serving as a readily available source of energy.
1.1.22 MMeellaannooccyytteess aanndd mmeellaannooggeenneessiiss
Although melanocytes comprise only a small proportion of the cells present in the epidermis of mammals, they are responsible for the production of the pigment melanin which accounts for virtually all of the visible pigmentation in their skin, hair and eyes. Melanin is pro- duced in specific and unique subcellular organelles, the so-called melanosomes. The functions of melanin are still discussed. It has a clear role in camouflage and sexual display.
But this function is more important for animals than for Homo sapiens. It is thought that in man the main functions of melanin are protection against UV light radiation and scavenging of radicals.
1
1..22..11 MMeellaannooccyytteess
Melanocytes are highly dendritic cells that originate from the neural crest. During embryogenesis they migrate through the developing body to three principal locations: the skin, the eyes and the hair bulbs.
Melanocytes in the eye are distributed in the choroid, iris and retina.
Eye melanocytes have very low rates of melanogenesis after fetal de- velopment and the synthesized pigment is not secreted, but remains in the melanocyte. Melanocytes in the eye are thought to function essentially as a photoprotective barrier.
Melanocytes that migrate to the skin are highly secretory. Within the hair bulbs the melanocytes produce melanosomes that are transferred to the hair shafts, thus giving the hair its visible color. The differ- ences in hair color (and also in skin color) in man are caused by dif- ferent types of melanin with differing visible colors that are produced by the melanocytes. The types of melanin produced and their distinct functional properties are discussed later in this chapter.
With age the melanocytes in the hair bulbs often become dormant and stop their melanin production, thus leading to the characteristic graying of the hair.
In skin itself the melanocytes are located at the junction of the der- mis and the epidermis in the stratum germinativum. The melanocytes have an extremely low mitotic rate in contrast to the surrounding cells. The melanosomes produced in melanocytes are transferred to the neighboring keratinocytes. The exact mechanism of this transfer is still debated.
The association of the melanocyte and its surrounding keratinocytes has often been defined as the „epidermal melanin unit“ where one melanocyte is normally associated with 36 keratinocytes.
As many of the precursors and intermediates in the melanin biosyn- thetic pathway are cytotoxic, the synthesis of the pigment takes place in specialized organelles named melanosomes. It seems that melanosomes are specialized members of the lysosomal lineage of organelles [1]. Melanosomes originate from the smooth endoplas- matic reticulum as a cytoplasmatic vesicle with an amorphous interior when analyzed by electron microscopy. At this point the granule is called a premelanosome and does not contain any of the enzymes needed for melanin synthesis. The main enzyme of melanogenesis, the so-called tyrosinase, and the other melanogenic determinants are synthesized on ribosomes. They are transported through the rough endoplasmatic reticulum to the Golgi apparatus where they are post- translationally processed and glycosylated. They are secreted within coated vesicles into the cytoplasmatic milieu and are transported specifically to premelanosomes. The enzymes are integrated in the melanosomal membrane with their catalytic centers facing inward.
During this process the amorphous structure of the melanosome changes into a characteristic fibrilar pattern along the longitudinal axis of the melanosome. Studies have shown that tyrosinase is cata- lytically competent while in transit through the Golgi apparatus. It is not clear how melanin synthesis is delayed until the enzyme arrives at the melanosome. It has been proposed that specific inhibitors are responsible for preventing the synthesis of melanin until incorpora- tion within the melanosome. There are four stages in the maturation of a melanosome: stage I, the “premelanosome” is a spherical organ- elle with matrix filaments that are not well defined; stage II, in which the typical elliptical melanosome is filled with a filamentous or laminar matrix; stage III is characterized by the deposition of elec- tron dense material on this matrix; and stage IV, with complete opacification of melanosomal contents by melanin deposition [1].
As this process of maturation of the melanosome proceeds, the or- ganelle is steadily moving away from the perinuclear region near the Golgi apparatus to the peripheral dendritic areas. Recent studies showed that the actin-based molecular motor Myosin V together with the protein Rab27a binds to melanosomes and participates in their transport to dendrites [2]. It was also shown that kinesin plays an important role in melanosome transfer. It is speculated that Myosin V together with Rab 27a is responsible for the transport of melano- somes in the cell cortex, while kinesin drives the melanosome trans- fer in the perinuclear region.
As mature melanosomes arrive at the end of the melanocyte dendrite, they are secreted in areas where the melanocytes intercalate with keratinocytes. The actual transfer of melanosomes into keratinocytes and the keratinocyte-melanocyte interactions during the transfer are not well characterized. Early light and electron microscopy studies suggested numerous possible mechanisms for melanosome transfer [3]. These include the release of melanosomes into the extracellular space followed by endocytosis, direct inoculation („injection“), keratinocyte-melanocyte membrane fusion and phagocytosis. Recent studies showed that the protease-activated receptor-2 (PAR-2) and lectins and their glycoconjugates play an important role in melano- some transfer [4].
Once melanosomes are transferred to keratinocytes, they are pack- aged in secondary lysosomes and arranged as supranuclear melanin caps above the keratinocyte nuclei [5]. As keratinocytes ascend to the epidermal surface from the basal and suprabasal layers where melanosome transfer takes place, melanosomes also ascend and are retained in the horny-layer cells for approximately two weeks. In Caucasian skin melanosomes show marked degradation and appear as melanin dust. In contrast, melanosomes from dark-skinned Negroid people show little degradation in the horny-layer cells [6].
1
1..22..22 MMeellaanniinn bbiioossyynntthheessiiss
There are basically two distinct forms of melanin in mammals. One group is the so-called eumelanins that are brown to black and insolu- ble. The other group is the pheomelanins that are reddish-brown and soluble in alkali. The initial steps in the synthesis of eumelanin and pheomelanin are controlled by the enzyme tyrosinase, which oxidizes the amino acid tyrosine to 3,4-dihydroxyphenylalanine (DOPA) (see Fig. 1.3).
Fig. 1.3 The biosynthetic pa thway of eumelanin and pheomela nin. (From Ito et a l . [ 7 ])
DOPA therefore can spontaneously autooxidize to dopaquinone with- out tyrosinase, but at slower rates than in presence of the enzyme.
Dopaquinone is an extremely reactive compound that, in the absence of thiols in the reaction medium, undergoes intramolecular cyclization leading to leukodopachrome and then to dopachrome. Dopachrome decarboxylates spontaneously to dihydroxyindole (DHI).
In the presence of divalent cations and the enzyme DOPAchrome tautomerase, also called tyrosinase related protein 2 (TRP2), the in- termediate 5,6-dihydroxyindole-2-carboxylic acid (DHICA) will result.
DHI is oxidized to indole-5,6-quinone and DHICA is oxidized to In- dole-5,6-quinone-carboxylic acid. It is speculated that the oxidation of DHICA is catalyzed by an enzyme called DHICA oxidase which is synonymous with tyrosinase related protein 1 (TRP1) and the oxida- tion of DHI by tyrosinase. The quinones are thought to build melanin by oxidative polymerization. Whether this polymerization step is un- der enzymatic control is not yet clear. It is thought that peroxidase or the melanocyte specific protein Pmel-17 play a role in this step of melanin synthesis [8].
Melanins generated in vitro from DHICA are brown in color, poorly soluble and of intermediate weight, whereas those generated from DHI are black, totally insoluble and of high molecular weight. These melanins are termed eumelanins. Eumelanins are a mixture of DHI- and DHICA melanins and on the individual basis, the chemistry of these pigments may vary to a considerable extent. Thus various forms of eumelanin that can be found in human skin interact differ- ently with UV light.
The other major diversion in the melanin biosynthesis pathway occurs upstream in the pathway immediately following the conversion of DOPA to dopaquinone. In the presence of sulfhydryl donors, probably cysteine, dopaquinone is converted to cysteinyl-DOPA.
Further oxidation, cyclization and polymerization leads to the forma- tion of pheomelanin. Pheomelanins have a yellowish-red color, are soluble in alkali and have a low molecular weight.
These different types of melanin are responsible for the differences in hair color in mammals and in man. Yellow to bright red hair result from the production of pheomelanin, whereas brownish to black hair have their origin in eumelanin production.
It is not clear, how the switch from eumelanin- to pheomelanin pro- duction or vice versa is controlled .
For mice it is known that the interactions of melanocyte stimulating hormone (MSH) and agouti signaling protein (ASP) play an important role [9, 10]. A black phenotype, originated by eumelanin production is elicited at conditions under which there is an overstimulation of the MSH receptor, whereas in contrast, conditions under which the function of the MSH receptor is abrogated or blocked by ASP result in the production of pheomelanin leading to a yellowish phenotype. To- day it is known that MSH and ASP function similarly in humans. It was shown that the phenotype of red hair and fair skin unable to tan is associated with mutations in the gene for the melanocyte- stimulating hormone receptor [11].
One can put the question why there exist different types of melanin.
Every type of melanin has its own physical and biological characteris- tics. DHI melanin is very good in photoabsorption, shows no photo- toxicity, but is highly cytotoxic. DHICA-melanins have reduced pho- tabsorption, no phototoxicity and are less cytotoxic. In contrast, pheomelanin provides only little photoabsorption, has a high photo- toxic potential, but low cytotoxicity. The optimum type of melanin must be a compromise between photoprotection while minimizing cyto- and phototoxicity.
1
1..22..33 RReegguullaattiioonn ooff mmeellaannooggeenneessiiss
Mammalian and therefore human pigmentation is a very complex process. This biosynthetic pathway is regulated at different levels by a variety of distinct factors. There are enzymes such as tyrosinase, TRP1 and TRP2 that regulate the synthesis of melanin.
These enzymes are also regulated (their activity, their production at the transcriptional and translational level, etc.). Keratinocytes pro- duce a lot of substances that play an important role in regulating melanocyte growth and differentiation. One of the major factors that affect human pigmentation is UV radiation.
One can divide skin pigmentation in two components. The first, con- stitutive pigmentation, is the pigmentation which is genetically de- termined in the absence of stimulatory influences, or in other words, the level of pigmentation in parts of the body that are not normally exposed to UV radiation. The second component, facultative pigmen- tation, is the level of pigmentation or tanning that occurs in response to stimulatory effects that are in man UV radiation and, to a certain degree, hormones.
These external stimuli are converted in intracellular messenger mole- cules that affect melanogenesis. Different signaling pathways mediat- ing melanogenesis will be discussed later in this chapter.
1.1.22..33..11 EEnnzzyymmeess iinn sskkiinn ppiiggmmeennttaattiioonn
There are three key enzymes known to date, all physiologically asso- ciated with melanosomes: tyrosinase, TRP1 and TRP2 (Tab. 1.1). Ty- rosinase, TRP1 and TRP2 are glycoproteins embedded in the melano- some membrane that share 70-80% nucleotide sequence homology with 30-40% amino acid identity. Among these three enzymes, ty- rosinase is the most critical and rate limiting enzyme.
Tyrosinase
Tyrosinase is the rate-limiting enzyme in the melanin biosynthesis pathway. It is a multifunctional copper-containing glycoprotein with a molecular weight de novo of 65 kDa and about 75 kDa when glycosy- lated [12]. It is an unusual enzyme as it catalyzes three distinct chemical reactions [13].
It catalyzes the first two steps in melanogenesis: the hydroxylation of tyrosine to DOPA and then the oxidation of DOPA to dopaquinone.
The third catalytic activity is the oxidation of DHI to indole-5,6- quinone. This function of the enzyme is still debated.
Another curiosity of tyrosinase is the fact that it requires DOPA as cofactor for the tyrosine hydroxylase reaction [14]. The product is therefore the cofactor for the synthesis of the product.
Rates of tyrosine hydroxylation in the absence of the cofactor are negligible. That raises the question where the initial DOPA cofactor derives from, since DOPA is not a normal amino acid available within the cell. This important question is still unresolved.
Tyrosinase is an extremely stable protein that is highly resistant to heat or proteases. It also has an unusually long biological half-life up to 10 hours in vivo.
Tyrosinase can be divided into three domains: an inner domain that resides inside the melanosome, a transmembrane domain and a cyto- plasmatic domain. The biggest part of the enzyme resides inside the melanosome and only 10% or 30 amino acids constitute to the cyto- plasmatic domain [15]. The inner domain contains the whole catalytic activity while the cytoplasmatic domain seems to play an important role in cellular trafficking of tyrosinase and in regulation of tyrosi- nase activity.
The transcription of the tyrosinase gene is regulated by a transcrip- tion factor called microphtalmia-associated transcription factor (MITF) [16]. MITF belongs to the basic-helix-loop-helix-zipper family and is known to interact with a specific DNA sequence termed M-box with the sequence GTCATGTGGCT that is present in the promoter re- gion of the tyrosinase gene [17]. There is evidence that the intracel- lular second messenger cAMP increases tyrosinase gene expression by enhancing the interaction between MITF and the M-box [18].
The regulation of the activity of tyrosinase is managed by phosphory- lation of the enzyme by protein kinase C-β [19].
The cytoplasmatic domain of tyrosinase contains two serine residues at amino acid positions 505 and 509 that are candidates for phos- phorylation by this serine/threonine kinase [15].
A recent study showed that PKC-β phosphorylates both serines but it is not clear whether phosphorylation of one or both of the serine residues is required for the activation of tyrosinase [20].
TRP1
The function of TRP1 is not clear yet. It has been proposed to be a lower specific activity tyrosinase [21], a dopachrome tautomerase [22], a tyrosine hydroxylase [23], a DOPA oxidase [24], a melano- somal catalase [25], or a DHICA oxidase [8]. More recently it has been suggested that TRP1 influences the activity of tyrosinase by stabilizing and/or forming a complex with tyrosinase [26]. In the pro- tein data base Swiss Prot the catalytic function of TRP1 is stated as
„Oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into In- dole-5,6-quinone-2-carboxylic acid. May regulate or influence the type of melanin synthesized“. The molecular weight of the precursor molecule is about 60 kDa. Synonyms for the enzyme are: DHICA oxi- dase, catalase B, glycoprotein 75 and melanoma antigen gp75. About the regulation of the transcription of the corresponding gene and the activity of the enzyme nothing is known.
TRP2
The function of TRP2 is that of a dopachrome tautomerase [27]. The molecular weight of the enzyme is disputed. The records range from 46 to 80 kDa.
The reason for these discrepancies in molecular weight is the fact that it is very difficult to purify TRP2 as it co-purifies with tyrosinase and may exist in a complex with tyrosinase, TRP1 and possibly other proteins.
T y r o s i n a s e T R P 1 T R P 2
S y n o n y m
M o n o p h e n o l m o n o o x y - g e n a s e
D H I C A o x i d a s e C a t a l a s e B G l y c o p r o t e i n - 7 5 M e l a n o m a a n t i g e n g p 7 5
D o p a c h r o m e t a u t o m e r a s e D o p a c h r o m e d e l t a -
i s o m e r a s e
S p e c i f i c i t y M e l a n o c y t e M e l a n o c y t e M e l a n o c y t e M o l e c u l a r w e i g h t 6 5 k D a (d e n o v o)
7 5 k D a ( g l y c o s y l a t e d )
6 0 k D a ( u n p r o c e s s e d p r e c u r s o r )
4 6 – 8 0 k D a ( i n d i s p u t e ) I s o e l e c t r i c p o i n t 4 . 3 U n k n o w n U n k n o w n
C a t a l y t i c a c t i v i t y
T y r o s i n e Æ D O P A D O P A Æ D o p a q u i n o n e
D H I Æ I n d o l q u i n o n e
D H I C A ÆI n d o l q u i n o n e
c a r b o x y l i c a c i d D o p a c h r o m e Æ D H I C A H a l f l i f e 4 – 1 0 h (i n v i v o) U n k n o w n U n k n o w n
M i s c e l l a n e o u s
H e a t - s t a b l e P r o t e a s e - s t a b l e C h e l a t o r - s e n s i t i v e
G l y c o s y l a t e d M e m b r a n e - b o u n d
D O P A c o f a c t o r - d e p e n d e n t
G l y c o s y l a t e d M e m b r a n e - b o u n d
H e a t - s e n s i t i v e P r o t e a s e - s e n s i t i v e C h e l a t o r - i n s e n s i t i v e
G l y c o s y l a t e d M e m b r a n e b o u n d
D O P A c o f a c t o r - i n d e p e n d e n t
Table 1.1 Characteristics of melanogenic enzymes
1.1.22..33..22 PPaarraaccrriinnee ccoonnttrrooll
Keratinocytes play an important role in the regulation of melanocyte growth and differentiation. They produce a variety of different fac- tors that act on melanocytes.
Melanocytes do not produce their own growth factors under normal conditions. There exist reports that one of the first steps in mela- noma development is the synthesis of autocrine growth factor basic fibroblast growth factor (bFGF) by melanocytes [28]. There is also evidence that α-MSH acts as an autocrine factor on melanocytes in melanogenesis [29].
Under normal conditions the proliferation of melanocytes is regulated by keratinocytes through the production of the main melanocyte growth factor bFGF. Other melanocyte growth factors include mast cell growth factor (MGF) and hepatocyte growth factor (HGF).
Their actions are not specific to melanocytes. HGF stimulates the growth of a variety of epithelial cells, and mast cell growth factor (synonymous with stem cell factor, steel factor or c-kit ligand) af- fects the proliferation of numerous cell types.
The most studied of the locally produced factors that regulate melanocytes are the cytokines and other inflammatory mediators. It has been suspected that some of these substances mediate the ef- fects of UV radiation and post-inflammatory pigmentary responses.
The cytokines interleukin 1 (IL-1), IL-6 and tumor necrosis factor-α (TNF-α) have been reported to inhibit both melanogenesis and melanocyte proliferation. These cytokines as well as TNF-β, IL-7 and interferon-γ (INF-γ) also induce the expression of intercellular adhe- sion molecule-1 (ICAM-1) and MHC class II molecules on cultured melanocytes. This could reflect a change in melanocyte function from a melanin producing cell to an immune competent cell.
Endothelins play an important role in melanocyte development, growth, melanogenesis, motility and dendricity. Studies showed that ET-1 affects melanocyte dendricity [30], proliferation [31], and melanogenesis [32], while ET-3 plays an important part in regulation of progenitor number and differentiation in melanocyte development [33]. To date ET-1 is the only mitogen that increases both melanin synthesis and melanocyte proliferation at very low doses in the nanomolar range [32].
1.1.22..33..33 HHoorrmmoonnaall ccoonnttrrooll
Melanocytes respond to many hormonal stimuli, and the two most im- portant groups are MSH peptides and sex steroids.
MSH peptides
There exist several MSH peptides. They are formed together with numerous other peptides including adrenocorticotrophic hormone
(ACTH), lipotrophins and endorphins from a precursor molecule called pro-opiomelanocortin (POMC) (see Fig. 1.4).
Fig. 1.4 The POMC fa mily of peptides (fro m Thody [12]) The co re sequences are shown as and as .
ACTH – adrenocorticotrophic hormone, CLIP – corticotrophic like intermediate lobe peptide, βEP – β-endorphin, JP – joining peptide, LPH – lipotrophin, MSH – melanocyte stimulating hormone, POMC – pro-opio melanoco rtin
The main site of POMC production is the pituitary gland but there is evidence that this protein and its cleavage products are also pro- duced in the skin [34, 35]. The pigmentary action of α-MSH in man was first demonstrated by Lerner and McGuire [36].
There exist several reports that α-MSH and related peptides fail to have an effect on melanogenesis in cultured human melanocytes [37]. There is strong evidence that this unresponsiveness of cultured melanocytes to MSH in vitro is related to mitogens normally used in melanocyte cell culture. If these mitogens are avoided, human melanocytes readily respond to MSH [38].
In mice the main effect of MSH is the increase of synthesis of eume- lanin.
From the MSH or melanocortin receptor seven different subtypes are known to date. Melanocytes express melanocortin receptor 1 (MC1R) [39].
The MC1R is a seven pass transmembrane G protein coupled receptor which activation leads to an increase in the second messenger cAMP.
In man at least 20 allelic variants of the MC1R have been described to date [11]. Four alleles are clear loss-of-function mutations that are causally associated with the development of red hair and cutane- ous sensitivity to UV radiation (tendency to burn) that is recognized as a major risk factor for melanoma skin cancer.
Recent studies showed that α-MSH is not the only POMC peptide with melanogenic activity and it is now established that ACTH binds to the MC1R and is also active in this respect [40]. There are reports that ACTH peptides are even more potent than α-MSH in stimulating melanogenesis and that ACTH1-17 is especially potent [29].
As it was said before, the major site of α-MSH production in mam- mals is the intermediate lobe of the pituitary. However this lobe is vestigial in adult humans and little if any α-MSH is produced there.
Now it is known that the molecule is produced in several cell types in the skin including keratinocytes and melanocytes and therefore the action that α-MSH has in the skin, is mediated by paracrine and/or autocrine mechanisms. The same applies to ACTH as it is produced by human keratinocytes.
Therefore these peptides are not hormones in the sense of the defini- tion of a hormone.
Steroids
Increased pigmentation of the nipples, areola, face, abdominal skin and genitalia is described for pregnant women. A study showed that sex steroids (diethylstilbestrol and estradiol) lead to a 1.2 to 2.5 fold increase of tyrosinase transcripts in cultured human melanocytes [41].
The amount of TRP1 transcripts was likewise enhanced but TRP2 transcripts were upregulated of about twenty fold over the initial transcript level.
This is a strong evidence that hyperpigmentation during pregnancy is mediated by a direct induction of melanogenesis by sex steroids.
1.1.22..33..44 TTrraannssmmeemmbbrraannee aanndd iinnttrraacceelllluullaarr ppaatthhwwaayyss
Several different signal transduction pathways operate in pigment cells, the best known being the adenylate cyclase/cAMP system that is activated by POMC derived peptides. The intracellular level of cAMP can also be upregulated by drugs or chemicals such as cholera toxin or isobutylmethyl xanthine (IBMX). It was shown that the action of cAMP is mediated by a cAMP-dependent protein kinase (PKA) which is a serine/threonine kinase. Activation of the cAMP pathway leads to an increase in tyrosinase gene transcription by enhancing the interac- tion of the specific transcription factor MITF with a specific DNA se- quence named M-Box that is located in the promoter region of the tyrosinase gene.
The second important pathway in human melanogenesis is the protein kinase C (PKC) pathway. In the last years experiments have shown that PKC-β is necessary to activate tyrosinase [20].
Using a pigmented human melanoma cell line that expresses PKC-β and an amelanotic subclone that has lost the expression of PKC-β but has equally abundant tyrosinase protein, it was shown that transfec- tion of PKC-β cDNA into amelanotic subclone cells activated tyrosi- nase.
It is thought that PCK-β activates tyrosinase by phosphorylation of the enzyme at the cytoplasmatic tail and also that the inactive form of PKC-β is activated through the intracellular messenger diacylglyc- erol (DAG).
DAG is produced together with 1,4,5-trisphosphate (IP3) from the precursor molecule phosphatidylinositol 4,5-bisphosphate (PIP2) that is cleaved by phospholipase C (PLC). IP3 interacts with IP3-sensitive
Ca2 + channels in the endoplasmatic reticulum, causing release of
stored Ca2 + that bind to calmodulin and activate a calmodulin de- pendent kinase (CaM kinase).
PLC can be activated by activated G proteins or by receptor tyrosine kinases that are discussed later in this chapter. To date it is known that ET-1 acts through the PLC/PKC pathway. But it is speculated, that the different signal transduction pathways are not isolated but that there exists a cross-talk between them. These speculations are supported by findings that ET-1 increases not only tyrosinase activity but also the intracellular cAMP level and the tyrosinase gene transcription.
The third important pathway is the tyrosine kinase pathway.
Receptor tyrosine kinases (RTKs) form a large and important class of cell-surface receptors whose ligands are soluble or membrane-bound peptide/protein hormones including insulin and growth factors. In the skin, growth factors act not like hormones in the definition of a hor- mone, they act as paracrine or autocrine factors. To date it is known that the growth factors b-FGF, MGF and HGF bind to tyrosine kinase receptors on the surface on human melanocytes [42].
Binding of the ligands to the receptor causes the RTK to dimerize and the protein kinase of each receptor monomer then phosphorylates a distinct set of tyrosine residues in the cytosolic domain of its dimer partner. This process is termed autophosphorylation.
A protein called Ras that belongs to the GTPase superfamily associ- ates with the activated domain of the RTK. Ras is a kind of an intra- cellular switch as it is in the on position or in its active form when GTP is bound, and in the off position or in the inactive form when GTP is hydrolyzed to GDP. The activated Ras binds to the N-terminal domain of a serine/threonine kinase called Raf. Raf binds to MEK and phosphorylates it.
MEK is a protein kinase that has a dual specificity as it phosphory- lates both tyrosine and serine residues. MEK phosphorylates and acti- vates MAP kinase that is another serine/threonine kinase. Activated MAP kinase translocates to the nucleus and phosphorylates the ter- nary complex factors, which initiates transcription of genes such as c-fos that is a very important transcription factor.
1.1.22..33..55 EEffffeecctt ooff UUVV rraaddiiaattiioonn
In man, sunlight is the most important physiological stimulator of the pigmentary system and is responsible for the tanning response. It is the UV spectrum of solar radiation that causes the tanning response in the skin. UV radiation can be divided into UVC (200-290 nm), UVB (290-320 nm) and UVA (320-380 nm). UVC is absorbed in the atmos- phere and does not reach the Earth’s surface. UVA and UVB have dif- ferent effects on human pigmentation. It is thought that UVA is re- sponsible for the immediate tanning that occurs within 24 hours and is based on oxidation of pre-existing melanin [43] and redistribution of melanosomes in the melanocyte from a perinuclear position to a more peripheral, dendritic distribution [44]. Immediate tanning is not very photoprotective against subsequent UV injury in contrast to the delayed tanning response.
The delayed tanning response, that begins as the immediate response fades out and progresses for at least 3-5 days after UV exposure, can be induced by both UVA and UVB, although the UVA induced response is of 2-3 orders of magnitude less efficient.
The delayed tanning response is accompanied by an increase in ty- rosinase activity [45]. UVA induced delayed tanning requires oxygen at the time of irradiation, whereas the UVB response does not.
It is thought that UV-induced DNA photodamage and/or its repair is at least one of the initial signals in stimulation of melanogenesis due to UV radiation [46].
Other signals are paracrine/autocrine factors that are produced by keratinocytes or melanocytes after UV stimulation.
1.1.33 FFaattttyy aacciiddss
1.1.33..11 NNoommeennccllaattuurree ooff ffaattttyy aacciiddss
Fatty acids are monocarboxylic acids with an acyclic and unbranched structure. Their basic formula is CH3[CH2]nCOOH where n can be any number from two to twentytwo. The total number of carbon atoms is in most cases even although odd-numbered carbon atoms containing fatty acids also exist.
Fatty acids can be classified according to their chain length and also according to their double bonds. Fatty acids that contain no double bonds are called saturated, with one double bond they are named monounsaturated and with two or more double bonds polyunsatu- rated. Depending on the number of double bonds from 2, 3, 4, 5 and 6 they are named dienoic, trienoic, tetraenoic, pentaenoic, and hex- aneoic.
The carbon atoms of the fatty acid can be numbered from carboxyl group to the terminal methyl group (∆ numbering system) or from the methyl group to the carboxyl side (W or n numbering system).
Palmitic acid, that is a saturated fatty acid, is abbreviated as C1 6:0.
Palmitoleic acid that contains one double bond is abbreviated as C1 6:1,∆9 or as C1 6:1,n-7 depending on the numbering system that is used.
The number after the ∆ or n signifies the position of the double bond relative to the carboxyl group or to the terminal methyl group, re- spectively.
1
1..33..22 BBiioossyynntthheessiiss ooff ppoollyyuunnssaattuurraatteedd ffaattttyy aacciiddss
Mammalian tissues contain four series of PUFA: n-9, n-7, n-6 and n-3 [47]. Interconversion between these families is not possible. The precursors of two of these groups are the monounsaturated fatty ac- ids palmitolieic and oleic acids, which can be synthesized endoge- nously from saturated fatty acids. The precursors for the other two groups are necessarily derived from dietary sources: linoleic and lino- lenic acid. All of the PUFAs found in mammalian tissues are synthe- sized from these four precursors by desaturation and elongation cata- lyzed by enzymes called desaturases and elongases.
1
1..33..33 PPUUFFAA iinn ccuuttaanneeoouuss bbiioollooggyy
From a series of papers published in 1929 and 1930 it was speculated that warm-blooded animals are not able to synthesize appreciable quantities of certain fatty acids [48, 49]. Rats maintained on a fat- free diet over a long period developed abnormalities such as growth retardation, severe scaly dermatosis and extensive water loss through the skin. These deficiencies could be reversed alone by the consump- tion of linoleic acid or linolenic acid and thus these fatty acids were heralded as essential fatty acids.
Essential fatty acid deficiency syndrome was uncommon in human adults and the first study in a human subject was done by the bio- chemist W. R. Brown, who himself went on a diet extremely low in fat for six months [50]. This experiment failed to develop any cutaneous or other visible abnormalities. With the appearance of parenteral nu- trition based on a system of continuous fat-free infusion, patients developed alopecia (baldness due to hair failure), brittle nails, des- quamating dermatitis, and increased susceptibility to infection.
The polyunsaturated fatty acids serve as cellular membrane phos- pholipid components which can influence the physicochemical charac- teristics of the lipid bilayer.
Changes in membrane lipids can modify the mobility and function of a variety of membrane proteins.
Linoleic acid is the most abundant PUFA in human skin. There is evi- dence that one functional role of linoleic acid is its involvement in the maintenance of the epidermal water barrier [51]. The physical structure of this water barrier is ascribed to sheets of stacked bilay- ers that fill the extracellular space of the uppermost layer of the epi- dermis. These lipid bilayers contain large amounts of linoleate rich sphingolipids.
The mechanism of the reversion of the symptoms of essential fatty acid deficiency (which include hyperproliferation and transepidermal water loss) by feeding with linoleic acid was elucidated in the last years at least for the symptom of hyperproliferation. In the epidermis linoleic acid is metabolized to 13-hydroxyoctadecadienoic acid (13- HODE) [52]. Studies showed that 13-HODE was incorporated into epi- dermal phosphatidylinositol 4,5-bisphosphate, resulting in the PLC catalyzed release of 13-HODE containing diacylglycerol [53]. It is thought that this molecule could modulate the activity of epidermal PKC and therefore influences epidermal hyperproliferation and differ- entiation.
The second important PUFA in the skin is arachidonic acid. It makes up to 9% of the total fatty acids in the epidermal phospholipids in human epidermis. It is the major metabolite of linoleic acid. Arachi- donic acid is metabolized via the cyclooxygenase pathway, predomi- nantly to the prostaglandins E2, F2a, and D2, and also via the 15- lipoxygenase pathway, predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) [54].
Prostaglandins play a central role in inflammation. 15-HETE was re- ported to improve the symptoms of psoriasis vulgaris after intrale- sional injection [55] and it was also reported to inhibit the 5- lipoxygenase activity and the generation of leukotriene B4 in neutro- phils and basophils in vitro [56]. These in vitro effects suggest an anti-inflammatory potential.
1
1..44 PPUUFFAA aanndd mmeellaannooggeenneessiiss
From the work of Shono [57] at the beginning of the eighties of the last century it is well known that fatty acids have an influence on the activity of tyrosinase of B16 mouse melanoma cells in vitro. In the last ten years this effect was examined in further work. Altogether four papers were published about this topic by Ando and his co- workers.
It was shown that the unsaturated fatty acids oleic acid, linoleic acid and α-linolenic acid lead to a decrease in melanin content and tyrosi- nase activity in cultured mouse melanoma cells of the cell line B16- F10 [58]. The growth rate of the cells was not altered by the sub- stances. In the same study it was shown that the fatty acids lighten ultraviolet-induced hyperpigmentation of the skin of guinea pigs. It was thought that this effect was caused by two distinct processes;
first the inhibition of the production of melanin and second the ac- celerated turnover of the stratum corneum.
It was also shown that linoleic acid can activate isolated PKC from B16 cells in vitro [59].
Linoleic acid does not alter the tyrosinase mRNA level in B16 cells [60] and has neither inhibitory influence on the activity of isolated tyrosinase. In a recent study a possible mechanism for the regulatory effect of polyunsaturated fatty acids on melanogenesis of cultured mouse melanoma cells was proposed [61]. It is thought that in the presence of polyunsaturated fatty acids in the medium the proteolytic degradation of tyrosinase is enhanced.
1
1..66 OObbjjeeccttiivveess ffoorr tthhee tthheessiiss
As previously mentioned, the inhibiting effect of unsaturated fatty acids on melanogenesis is already well known. It is proven that the addition of oleic, linoleic or linolenic acid to the medium of cultures of B16 mouse melanoma cells leads to a decreased intracellular melanin content [58, 61]. In contrast addition of saturated fatty ac- ids to the medium leads to an increase in intracellular melanin con- tent. In both cases the melanin content correlates with the activity of tyrosinase, the main enzyme of melanin biosynthesis.
To date the inhibiting effect of unsaturated fatt acids on melano- genesis was shown in an in vitro mouse melanoma cell culture model and in vivo in an animal model (guinea pig). In the animal model it was shown that unsaturated fatty acids can lighten UV-induced tan- ning but it is not known if they have an influence on constitutive pigmentation, which is defined as the pigmentation which is geneti- cally determined in the absence of stimulatory influences.
As cell culture model B16 mouse melanoma cells were used. From this cell line different sublines exist. The American cell bank ATCC lists 3 sublines that are named B16-F0, B16-F1 and B16-F10. The European cell line collection ECACC further lists a cell line named B16 mela- noma 4A5. Therefore it seems that four sublines of the B16 line exist.
These sublines are not well characterized and nothing is known about the differences between them, although B16 cells are often used in studies investigating melanin biosynthesis.
To date there exist neither in vitro nor in vivo studies of the influ- ence of fatty acids on melanogenesis of normal human melanocytes.
In first experiments with B16-F1 mouse melanoma cells and linoleic acid we were not able to detect an inhibition in tyrosinase activity.
Thus in this work the following questions should be answered:
What is the function of linoleic acid, linolenic acid (synonym α- linolenic acid), arachidonic acid and γ-linolenic acid on melanogenesis of B16-F10 cells and B16-F1 cells?
Is there a difference between the two sublines of the B16 mouse melanoma cell line in the reaction on the incubation with polyunsatu- rated fatty acids ?
Do polyunsaturated fatty acids also affect the melanin synthesis of cultured normal human melanocytes?
Which effects have polyunsaturated fatty acids in a pigmented 3D in vitro skin model?
2.2. MMaatteerriiaall aanndd MMeetthhooddss 2.2.11.. CCeellll ccuullttuurree
2.2.11..11 IIssoollaattiioonn aanndd ccuullttiivvaattiioonn ooff nnoorrmmaall hhuummaann mmeellaannooccyytteess
Normal human melanocytes were isolated from human foreskins and cultured according to the method of Eisinger [62] with few modifica- tions.
Culture media and solutions
PBS without Ca2 + and Mg2 + (PBSA)
137 mM NaCl [Merck 6404] 8.00 g/liter 2.68 mM KCl [Merck 4936] 0.20 g/liter 1.47 mM KH2PO4 [Merck 4873] 0.20 g/liter 8.09 mM Na2HPO4 * 2H2O [Merck 6580] 1.44 g/liter
PBS-EDTA
137 mM NaCl [Merck 6404] 8.00 g/liter 2.68 mM KCl [Merck 4936] 0.20 g/liter 1.47 mM KH2PO4 [Merck 4873] 0.20 g/liter 8.09 mM Na2HPO4 * 2H2O [Merck 6580] 1.44 g/liter 0.537 mM EDTA [Sigma ED2SC] 0.20 g/liter
0.25 % Trypsin
2.5% Trypsin [Gibco25090-028] was diluted with PBSA.
Trypsin-EDTA
2.5% Trypsin [Gibco25090-028] was diluted with PBS-EDTA to a con- centration of 0.05%.
Complete medium
MEM with NEAA [Gibco 41500-067]
100 units/ml penicillin [Gibco 15140-122]
0.1 mg/ml streptomycin [Gibco 15140-122]
0.25 µg/ml Fungizone [Gibco 15290-018]
Washing medium
MEM with NEAA [Gibco 41500-067]
1000 units/ml penicillin [Gibco 15140-122]
1 mg/ml streptomycin [Gibco 15140-122]
2.5 µg/ml Fungizone [Gibco 15290-018]
Growth medium
Complete medium without Fungizone supplemented with:
5% FBS [Gibco 10091-148]
16 nM PMA [Sigma P 8139]
2.5 nM cholera toxin [Sigma C 8052]
0.1 mM IBMX [Sigma I 7018]
Method
Human foreskins from circumcisions were collected in complete me- dium and stored near the surgical area at 4°C. Specimens were deliv- ered to the cell culture laboratory the same day. The skin was trans- ferred to a laminar flow hood where the following preparations were carried out under aseptic conditions. The skin was immersed in wash- ing medium for 5, 10 and 15 min, each time in fresh solution. This was followed by a wash with complete medium. After that the skin was transferred to a Petri dish epidermal side down, and using curved scissors, fat and connective tissue were removed. Then the skin was cut into small pieces of about 25 mm2. The pieces were washed with PBSA, placed in 0.25% trypsin and kept at 4°C overnight (~16 h).
Following this incubation each piece of skin was removed from the trypsin solution, placed on a Petri dish (dermal side down) and held with forceps.
Using a second pair of forceps epidermis was peeled off and collected in trypsin/EDTA solution. A single cell solution was prepared by pi- petting. The solution was transferred to a tube containing a small amount of FBS. The mixture was centrifuged for 10 min at 180 x g.
The pellet was resuspended in growth medium, filtered through a 100 µm cell strainer [Falcon 2360] and the resulting cell suspension was seeded into cell culture dishes. Cells were cultured in a CO2 in- cubator [Heraeus] at 37°C and 5% CO2 . Medium was changed 48 h later to remove unattached cells. Afterwards medium was changed twice a week and the cells were subcultured when confluence reached about 70%.
2
2..11..22 EElliimmiinnaattiioonn ooff ffiibbrroobbllaasstt ccoonnttaammiinnaattiioonn
Contaminating fibroblasts were eliminated by differential trypsiniza- tion or by selective destruction of fibroblasts with geniticin.
a) Differential trypsinization
Medium was discarded and cells were washed with PBSA. After that 0.25% trypsin solution was added. The solution was removed after it had wet the surface of the cell culture dish. The cells were observed under the inverted microscope [Olympus]. Melanocytes detached faster from the surface than fibroblasts and were collected in growth medium and replated at a density of 1 x 104 cells/cm2. If there were still contaminating fibroblasts, the procedure was repeated. Cells were cultured at the conditions described above and subcultured when confluence was reached.
b) Selective elimination with geneticin
If the method above did not succeed in eliminating the fibroblast contamination, the cells were selectively killed by treatment with ge- neticin. This method was described by Halaban et al. [63].
They used geneticin at a dose of 100 µg/ml to selectively kill fibro- blasts in mixed cultures of melanocytes and fibroblasts. We treated contaminated cultures with a dose of 200 µg/ml geneticin for seven days as a dose of 100 µg/ml did not succeed in eliminating all fibro- blasts. Melanocytes were not affected by this treatment.
2.2.11..33 CChhaarraacctteerriizzaattiioonn ooff mmeellaannooccyytteess
Melanocytes were identified by immuncytochemistry. Tyrosinase was specifically detected with an anti tyrosinase antibody that was visual- ized with a second antibody labeled with fluorescein.
Materials
•Cultures of normal human melanocytes
•Cultures of normal human fibroblasts
•Culture slides [Falcon]
•PBSA
•Growth medium for melanocytes
•Growth medium for fibroblasts:
DMEM [Gibco 31600-026]
10% FBS [Gibco 10091-148]
100 units/ml penicillin [Gibco 15140-122]
0.1 mg/ml streptomycin [Gibco 15140-122]
•Acetone [Merck]
•PBS-Tween:
PBSA including 0.02% (w/v) Tween 20 [Bio-Rad 170-6531]
•Antibody buffer:
PBSA including 1% (w/v) BSA [Sigma A-8551]
•Antibody I:
Tyrosinase Ab-1 (T311) [NeoMarkers MS-800-P1]
•Antibody II:
Goat anti mouse IgG fluorescein labeled [Pierce 31569]
•PolyDABCO:
0.3 g Tris HCl [Sigma T-3253]
2.2 g Tris base [Sigma T-1503]
20.0 ml H2O bidest.
2.0 g Polyvinyl alcohol [Sigma P-8136]
4.5 ml Glycerin [Merck 4094]
0.75 g DABCO [Fluka 33480]
Tris HCl and Tris base were solubilized in 20 ml H2O. Polyvinyl alco- hol was added in portions to the solution under stirring. The solution was stirred for 30 min. Then glycerin was added and the solution was warmed until glycerin had dissolved and the solution became clear.
Afterwards the solution was cooled down to RT and pH was set to 8.70 with 2 M HCl. The solution was stored at 4°C.
Method
The cells were cultured in culture slides in a humidified incubator at 37°C and 5% CO2 for four days. The medium was discarded, the me- dia chamber removed and the cells were washed with PBSA three times. After this the cells were fixed for 10 min with ice cold acetone on ice. After this fixation the slides were washed three times with PBS-Tween. Antibody I was diluted 1:100 with antibody buffer and 100 µl of the solution were pipetted on the culture slide. The culture slides were incubated for 45 min at RT in a humid chamber. After this incubation the slides were washed with PBS-Tween three times. Anti- body II was diluted 1:50 with antibody buffer.
On each slide 100 µl of antibody solution were pipetted. The slides were incubated at RT in the dark in a humid chamber. After 45 min the slides were washed three times with PBSA and mounted with PolyDABCO. The specimens were examined under a fluorescence mi- croscope [Olympus BX50] using an exciter filter BP460-490 [Olympus]
and a barrier filter BA515IF [Olympus]. Photographs were taken using a reflex camera [Olympus OM-4 Ti] and 35 mm film [Kodak].
2.2.11..44 CCuullttiivvaattiioonn ooff BB1166--FF11 aanndd BB1166--FF1100 mmoouussee mmeellaannoommaa cceellllss
B16-F10 (CRL-6475) and B16-F1 (CRL-6323) mouse melanoma cells were purchased from ATCC. These cell lines were isolated from the mouse strain C57BL/6J and deposited at ATCC by the Naval Biosci- ences Laboratory. The following cell line descriptions were given by ATCC:
B16-F1 Organism: Mus musculus (mouse)
Tissue: melanoma
Strain: C57BL/6J
Morphology: fibroblast-like
Depositors: Naval Biosciences Laboratory Tumorigenic: yes, in syngenic mice
Note: This cell line produces melanin which may cause the culture medium to turn brown/black. This is normal and should not be misinterpreted as contamination
B16-F10 Organism: Mus musculus (mouse)
Strain: C57BL/6J
Depositors: Naval Biosciences Laboratory
The growth medium consisted of:
DMEM [Gibco 31600-026]
5% FBS [Gibco 10091-148]
100 units/ml penicillin [Gibco1 5140-122]
0.1 mg/ml streptomycin [Gibco1 5140-122]
The cells were grown in cell culture Petri dishes in a humidified CO2
incubator at 37°C and 7% CO2. Due to the rapid growth cells were subcultured twice a week on Monday and on Friday. Medium was changed on Wednesday. For subculturing the medium was discarded, the cells were washed with PBSA and little 0.25% trypsin solution was added. After it had wet the surface the solution was removed and the cells were incubated at 37°C for 5 min. After this incubation the cells had detached from the surface and were collected in culture medium. The cells were seeded at a density of ~1.7 x 103/cm2.
2.2.11..55 DDeetteerrmmiinnaattiioonn ooff cceellll nnuummbbeerr
The cell number was determined using a cell counter CASY 1 [Schärfe System]. For counting, monolayers had to be trypsinized and resuspended in medium.
Material
•Cell monolayer
•PBSA
•0.25% Trypsin
•Growth medium
•CASYton [Schärfe System]
•Counting cups [Schärfe System]
•CASY 1 [Schärfe System]
Method
The cell suspension was diluted 1:100 in 10 ml CASYton counting fluid in a disposable counting cup. The suspension was mixed well and the cell number was determined according to the manufacturer’s instructions.
2
2..22 TTeesstt ssuubbssttaanncceess
The following substances were chosen to investigate the influence on melanogenesis of normal human melanocytes and mouse melanoma cells:
Linoleic acid (all cis-9,12-Octadecadienoic acid) [Sigma L-1012]
Arachidonic acid (all cis-5,8, 11,14-Eicosatetra enoic acid) [Matreya 1042]
α-Linolenic acid (all cis-9,12,15-Octadecatrieno ic acid) [Matreya 1024]
γ-Linolenic acid (all cis-6,9, 12-Octadecatrieno ic acid) [Matreya 1153]
The test substances were stored under nitrogen at –20°C to reduce oxidation of the unsaturated bonds.
Preparation of stock solutions
Stock solutions (50 mM) [Merck 1.00983] were prepared in ethanol and stored under nitrogen at –20°C.
2.2.33 DDeetteerrmmiinnaattiioonn ooff ccyyttoottooxxiicciittyy
Cytotoxicity of the test substances was determined using the neutral red uptake (NRU) method [64].
2
2..33..11 CCyyttoottooxxiicciittyy oonn nnoorrmmaall hhuummaann mmeellaannooccyytteess Material
•Free fatty acid free FBS:
Free fatty acid free fetal bovine serum was prepared using three ex- tractions with equal volumes of diethyl ether [Merck 1.00921] in a separating funnel, according to the method of Ando [58]. The resid- ual ether was removed using an evaporator [Büchi].
•Test medium for NHM:
MEM with NEAA [Gibco 41500-067]
10% free fatty acid free FBS
100 units/ml penicillin [Gibco 15140-122]
0.1 mg/ml streptomycin [Gibco 15140-122]
16 nM PMA [Sigma P 8139]
0.1 mM IBMX [Sigma I 7018]
•NR-medium
Test medium containing 50 µg/ml neutral red [Sigma N-4638]. NR- Medium was prepared 24 hours before use and incubated overnight at 37°C. Medium was centrifuged for 10 min at 1000 x g to spin down precipitated dye.
•NR-fixation solution
1% (v/v) acetic acid [Merck 1.00063], 50% (v/v) ethanol [Merck 1.00983], 49% (v/v) H2O bidest. The solution was stored at RT.
•Cultures of NHM
•96 well cell culture plates [Sarstedt]
•Deep well plate [Eppendorf]
•Multipette plus [Eppendorf]
•Multichannel pipette [Eppendorf]
•Test substances
